Using the recently developed TAR (Transformation-Associated Recombination) cloning technique, it is possible to directly isolate specific chromosomal regions and genes from complex genomes as large linear or circular YACs using a complete or a partial sequence information. During last year a work was focused on further optimizing of TAR cloning to make the technology more widely used in the scientific community. In addition, entire copies of four human genes including metastasis-suppressor gene KAI1, a paternally expressed gene Ubiq and the telomerase reverse transcriptase hTERT gene were successfully isolated for further functional studies. TAR cloning technique has been also applied for isolation and characterization of centromeric regions of human chromosome. As a part of this study a complete sequence of centromeric region of the human Y chromosome was determined. This centromeric fragment containing ~140 kb of alphoid DNA was used for construction of small circular Human Artificial Chromosome lacking telomeres (HAC). When transfected into human cells, it is stably maintained at 1-2 copies per cell. Development of the HAC cloning system provides new opportunities for human gene therapy and could have profound effects on our understanding of mechanisms of human chromosome segregation.